WO2004071524A1

WO2004071524A1 - Albumin solution and method for the production thereof

Info

Publication number: WO2004071524A1
Application number: PCT/EP2004/001397
Authority: WO
Inventors: Werner Gehringer; Katharina Pock; Jürgen Römisch; Tor-Einar Svae; Christoph Kannicht
Original assignee: Octapharma Ag
Priority date: 2003-02-13
Filing date: 2004-02-13
Publication date: 2004-08-26
Also published as: RU2005128507A; CY1106793T1; EP1592439B1; PT1592439E; AU2004212324A1; CN1798573A; UA80469C2; JP2006517938A; DK1592439T3; PL376644A1; AU2004212324B2; CN100384471C; MXPA05008276A; EP1592439A1; IL169828A0; RS20050624A; DE502004003834D1; BRPI0407458A; ATE362376T1; RU2305556C2

Abstract

The invention relates to a therapeutically usable virus-inactivated albumin and a method for producing a therapeutically usable virus-inactivated albumin, which is characterized by a combination of the following steps: (a) a first aqueous albumin solution is subjected to a virus inactivation treatment according to the SD method by contacting said aqueous albumin solution with SD reagents at a temperature of less than 45 °C; (b) the SD reagents are at least essentially removed by means of oil extraction and subsequent hydrophobic interaction chromatography, a hydrophobic matrix, especially a matrix to which optional hydrophobic groups can be bonded, being used for the chromatography process provided that said groups are aliphatic groups wherein C > 24, and a second albumin solution is obtained to which (c) one or several stabilizers from the group comprising sugar, amino acids, and sugar alcohols is/are optionally added provided that no indole stabilizer and no C6-C10 fatty acid are used as stabilizers; whereupon (d) the second albumin solution, to which stabilizer has been optionally added, is finished and sterilized by filtration and optionally filled into final containers.

Description

Albumin solution and process for its preparation

The invention relates to a therapeutically usable virus-inactivated albumin and a method for its production.

Albumin is the most strongly represented plasma protein in the blood plasma. Albumin can bind many endogenous and exogenous substances to its molecule. One of its main functions is based on this binding capacity: the transport of the substances bound to albumin.

Due to this binding capacity, albumin is also an important depot for a variety of compounds, such as long chain fatty acids, bilirubin, tryptophan, thyroxine or metal ions. Active pharmaceutical ingredients such as warfarin, digitoxin or naproxen are also bound to albumin and transported.

In this context, however, it is crucial to know that only the free portion of the active pharmaceutical ingredient, i.e. the one not bound to albumin is the one that has the pharmacological effect. A reduction in the portion bound to albumin increases the free portion and thus the pharmacological activity.

All of the commercially available albumin preparations are produced by means of a modified Cohn fractionation, this method generally consisting of several fractionation steps. Pasteurization (10 hours at 60 ° C) of the albumin concentrate has been used as a virus inactivation step for decades. Stabilizers are added to avoid denaturing albumin during this step. According to the European Pharmacopoeia, sodium caprylate (sodium octanoate) or N-acetyltryptophan or a combination of both are used as stabilizers.

BESTATIGUNGSKOPIE To obtain virus-inactivated factor VIII preparations or other plasma proteins, the so-called SD method is used, as described, for example, in EP-A-0 131 740. Reference is expressly made to this publication.

Based on his own studies, the applicant is aware that the binding capacity of commercially available albumin. compared to natural albumin. This is attributed to the fact that the stabilizers used in pasteurization are bound by the albumin and in this way occupy important transport points, which reduces the binding capacity. This means that patients who receive such albumin preparations will receive a significantly increased concentration of free, i.e. are not exposed to the active substance bound to albumin, which naturally entails an increased risk of excessive pharmacological effects and side effects for the patient.

The object of the present invention is to provide an albumin preparation which does not have this disadvantage.

1 shows the UV absorption behavior of albumins of different provenance as a function of the elution time during the chromatographic separation.

Figure 2 illustrates the binding behavior of albumins of different origins in the presence of different concentrations of phenylbutazone.

Fig. 3 shows the binding behavior of various ^albumins' illustrated provenance in the presence of various concentrations of warfarin.

The problem is solved by a therapeutically usable virus-inactivated albumin with an increased binding capacity for substances compared to pasteurization-inactivated albumin. The In particular, albumin according to the invention has a binding capacity which is at least 10% higher than albumin virus-inactivated by pasteurization, typically 20 to 500% increased binding capacity, in particular 100% to 500% increased binding capacity. Depending on the substance to be bound, even higher values are possible in individual cases.

The substances are, in particular, those which are bound and / or transported by native albumin, which in particular includes low molecular weight active substances. In particular, the low molecular weight active ingredients are organic or inorganic substances, nucleic acids, polypeptides, which typically have a molecular weight of < _. Have 10,000 da.

For the therapeutic purposes mentioned, the albumin according to the invention can be in a liquid solution or in a solid state, in particular in a lyophilized form.

The albumin according to the invention is also obtainable by a process which is characterized by the combination of the following steps:

(a) subjecting a first aqueous albumin solution to a virus inactivation treatment according to the SD method by contacting with SD reagents at a temperature below 45 ° C.,

(b) Removal of the SD reagents by oil extraction and subsequent hydrophobic interaction chromatography, at least essentially, wherein a hydrophobic matrix, in particular a matrix to which hydrophobic groups can optionally be bound, is used for the chromatography, with the proviso that these groups have aliphatic groups C> 24, and a second albumin solution is obtained which

(c) optionally with one or more stabilizers from the group consisting of sugars, amino acids and sugar alcohols, with the proviso that no indole stabilizer and no C ₆ -C0 ₀ - fatty acid is used as the stabilizer, after which (d) the second albumin solution, optionally mixed with stabilizer, is finished and sterile filtered and, if appropriate, filled into final containers.

The term indole stabilizer is intended to encompass all stabilizers which have an indole structure, e.g. N-acetyl tryptophan.

The SD (= solvent / detergent) method for inactivating viruses is known from EP-A-0 131 740. In this publication, albumin is also mentioned among other proteins.

It is known from EP-A-0 366 946 that the SD reagents can be removed with vegetable oils, for example soybean oil, and subsequent hydrophobic interaction chromatography. To the extent that it overlaps with the method according to EP-A - 0 366 946, the method according to claim 8 is therefore to be regarded in one aspect as an analogy method for the production of the albumin according to the invention. For chromatography, however, a matrix is preferably proposed there, for example a silica matrix, to which hydrophobic side chains, namely branched or unbranched C ₆ -C ₂₄ alkyl chains, are bound.

It has surprisingly been found that the use of a hydrophobic matrix instead of a matrix which carries, for example, C ₁₈ alkyl chains as hydrophobic side chains, has a higher binding capacity for adsorbing detergents. Accordingly, no further hydrophobic groups need be bound to the matrix used according to the invention. A method using such a matrix is therefore also the subject of the invention.

Virus inactivation is advantageously carried out at a temperature in the range from 25 to 40 ° C.

A preferred embodiment of the method according to the invention consists in that the virus inactivation is carried out over a period in the range from 4 to 6 hours. Glycine is a very good stabilizer.

Castor oil is very suitable for oil extraction.

It has proven to be particularly advantageous for the cleaning effect if a polystyrene-divinylbenzene polymer or a methacrylate-based polymer is used as the hydrophobic matrix.

The hydrophobic matrices used according to the invention can carry branched or linear aliphatic groups with more than 24 carbon atoms.

Depending on the starting material used, a step to deplete the so-called precallikrein activator activity (PKA) may be required. PKA is known to cause blood pressure to drop after administration of preparations containing PKA by releasing the vasoactive substance bradykinin from the high molecular weight kininogen (HMWK).

PKA is usually inactivated during the pasteurization of protein preparations. Since heat treatment, by which experience has shown that the PKA is at least partially inactivated, is disadvantageous for the albumin produced according to the process for the reasons mentioned above, PKA can be removed by special measures if necessary. This includes incubation with activated carbon with subsequent filtration, preferably with depth filters, or direct filtration through filters containing activated carbon.

Ion exchangers, such as cation or anion exchangers, are also well suited for removing the PKA. This can be done by contacting the albumin-containing solution with the matrix in columns, or batch processes familiar to the person skilled in the art. Alternatively, dextran sulfate or heparin matrices can be used to reduce PKA.

The PKA is reduced in the albumin-containing solution obtained, in the ideal case it is no longer detectable. According to the current state of the art, PKA is identical to the activated (coagulation) factor XII (FXIIa), which is generated from its proenzyme form (FXII). This can be done on surfaces autocatalytically or by enzymatic action, for example Kallikreins. Accordingly, depletion of the FXII (proenzyme) is recommended as the starting substance of the PKA, but is not absolutely necessary. However, in order to prevent renewed generation of PKA from the proenzyme form, this can also be removed by ion exchange chromatography. The depletion of the FXII can optionally be carried out in order to enable long-term storage of the albumin in the liquid state. This is also important after thawing an albumin solution that may be stored in a frozen state. Accordingly, the albumin solution can be frozen after filling into the final containers, but can also be cooled in a liquid or freeze-dried state and stored at a temperature of up to 40 ° C.

To remove the PKA or PKA precursor substances, any precallikrein activator activity (PKA) that may be present before or after steps (a), (b) or (c) can be removed in a manner known per se, the albumin solution in particular

A) is brought into contact with activated carbon, after which the activated carbon is removed from the albumin solution, or

B) is subjected to ion exchange chromatography.

Step (A) is carried out at an albumin concentration between 1 and 25% by weight, in particular between 5 and 10% by weight.

Step (B) is carried out in particular at an albumin concentration between 5 and 10% by weight.

A further embodiment of the method according to the invention consists in that the ion exchanger is an anion exchanger and the albumin solution is buffered with sodium acetate in the range from 100 to 150 mmol / l and the pH in the range from 5.0 to 6.0, in particular <5.5 lies.

Furthermore, a method is described which is characterized in that the ion exchanger is a cation exchanger and the Albumin solution is buffered with sodium acetate in the range of 20-30 mmol / l and the pH is in the range of 4.8-6.0, in particular in the range of 4.8-5.2.

The invention relates. furthermore an albumin solution which can be obtained by the process according to the invention. This method is applicable to albumin solutions obtained from various sources, e.g. from blood plasma or serum, from albumin-containing fractions from plasma fractionation, from albumin obtained from culture supernatant after recombinant production or transgenically produced albumin or from the medium containing the albumin, such as milk.

A preferred embodiment of the invention is described in more detail using the following example.

example

1000 g of an aqueous albumin solution from the Cohn process (after dia / ultrafiltration) with a protein content of about 23%, Triton X-100 and tri-n-butyl phosphate (TNBP) are added to a concentration of 1% each. The albumin solution is then stirred at 30 ° C. for 4 hours.

To remove the SD reagents, castor oil is first added with stirring to a concentration of 5%, while the solution is brought to a temperature in the range from 20-25 ° C. The mixture is then stirred for 30 minutes. After stirring, the 60 minute mixture is left to stand, whereby a heavy, aqueous phase and a light phase form. The heavy phase is separated off and filtered through a filter with membranes with a pore size of <1 μm and <0.45 μm. The light phase (oil phase) contains the TNBP and is discarded.

The filtered solution is passed through a solid phase extraction column to separate the Triton X-100. A polystyrene-divinylbenzene polymer (Amberchrome CG 161) without hydrophobic side chains is used as the hydrophobic matrix. Water for injection is used to wash out the column, this process being monitored by measuring the UV absorption at 280 nm. The column is regenerated after use.

The following can be added as stabilizers: glycine, glutamate, arginine, maltose, sorbitol or mixtures of the substances.

The solution obtained is brought to pH = 7.0 and the protein content is adjusted to 200 g / l and the sodium content to 80 mmol / l Na ⁺ . The solution is then sterile filtered through a membrane filter with a pore size of <0.2 μm.

The sterile-filtered solution is filled into sterile, pyrogen-free PVC bags under aseptic conditions and labeled.

The labeled bags are frozen at a temperature of <-60 ° C so that the temperature inside the bags reaches <-30 ° C. The bags are stored at this temperature (< _. -30 ° C).

Präkallikreinabreicherung

In the case of PKA depletion, the following variants can be used: a) An albumin solution with a protein concentration of 1-25% by weight, in particular 5-10% by weight, is added with 3-10% by weight, in particular 5 % By weight activated carbon stirred at pH = 5 for one hour. The activated carbon is then filtered off. b An albumin solution with a protein concentration of 5-10% by weight is subjected to ion exchange chromatography (DEAE-Sepharose, Q-Sepharose) at pH 5-6, in particular <5.5, in a 100-150 mmol sodium buffered system , Due to the high ionic strength, a PKA-free albumin solution is obtained in one pass. c) An albumin solution with a protein concentration of 5-10 wt .-% is at pH 5-6, preferably 4.8-5.2, in a 20-30 mmol / l sodium acetate buffered system of an ion exchange Chromatography (SP-Toyopearl, CM-Sepharose) subjected. A PKA-free albumin solution is obtained in one pass.

Final Formulation

The solutions obtained are each brought to pH = 7.0 and the protein content is adjusted to 200 g / l and the sodium content to 80 mmol / l Na ⁺ . The solution is then sterile filtered through a membrane filter with a pore size of <0.2 μm.

The sterile-filtered solutions are filled into sterile, pyrogen-free PVC bags and labeled under aseptic conditions.

The labeled bags are frozen at a temperature of <-60 ° C so that the temperature inside the bags reaches <-30 ° C. The bags are stored at this temperature (< _. -30 ° C) _. Measurement of the binding of substances to different albumin preparations

A direct method for determining the binding properties of substances to albumin is offered by size exclusion chromatography (SEC) according to Hummel and Dreyer (Biochim Biophys Acta 1962; 63: 530-532).

To do this, an SEC column is equilibrated with a buffer solution containing the binding ligands (eg phenylbutazone or warfarin). The absorption in the UV range is monitored continuously. The protein is applied to the column and eluted in the equilibration buffer. Bound ligand elutes together with the albumin, the unbound, usually smaller ligand, elutes accordingly later. The absorption of the bound ligand usually interferes with the absorption of the albumin and possible accompanying substances such as stabilizers. The later "negative" so-called "vacancy" peak is caused by the depletion of the ligand in the subsequent buffer, which takes up more area the more bound to the previously eluted albumin. Koizumi et al. (Biomed Chromatogr 1998; 12: 203-210) used this method in a slightly modified form to investigate the binding capacities of substances on albumin or its affinities, for example by adding constant concentrations of albumins in separate runs with increasing amounts of the ligand and thus the binding capacity in the form of albumin-to-substance ratios could be determined.

A Biosep-SEC-S 4Ö00 column, 300x4.6 mm micron (Phenomenenx) on a Shimadzu HPLC system was used for this investigation. The buffer flow rate was 0.35 ml / min, the column having been equilibrated with 50 mM potassium phosphate buffer, pH 7.4. The protein concentration was 50 μM, the injection volume 80 μl. Phenylbutazone was tracked at 263 nm, warfarin at 308 nm. The linear absorption ranges had previously been determined.

The albumin described in this application was used (1) and two commercially available (stabilized) albumin preparations (2,3). These were 20% albumin solutions.

FIG. 1 shows a superposition of four different chromatograms, the column being equilibrated in 50 μM phenylbutazone (in phosphate buffer). With a retention time of 11 minutes, the albumin initially eluted, the peak indicating the sum of the protein absorption and that of the bound substance. At 14.5 min, an N-acetyl-tryptophan (stabilizer) peak is usually seen in the case of a commercial albumin. After 18.5 min the 'vacancy' peak appears in the form of a 'negative' absorption representation relative to the level of the equilibration buffer including the substance. The higher (in the negative sense) this peak or the larger the peak area, the more substance has bound to the previously eluted albumin.

2 shows the UV absorptions of three phenylbutazone concentrations bound to albumin (after subtraction of the buffer peak). For this purpose, two commercially available albumins (containing caprylate and N-acetyl-tryptophan) and the albumin prepared by the process described in this application were chromatographed and the binding qualities compared. At comparable molar concentrations of phenylbutazone to albumin, it is clear that the peak in area and height are clearly larger in the case of the new albumin. This applies similarly to the second example, namely warfarin, as shown in FIG. 3.

These results underline that the commercial albumin is inferior in binding properties to the albumin described here.

Comparison of the binding capacity of RP-18 columns compared to polystyrene-divinylbenzene polymers (Amberchrome 161 M).

Test system: column volume: 44 ml

Flow rate: 4 ml / min

The column was loaded with a 1% Triton X-100 solution. The Triton X

Eluate content measured after each column volume using reverse phase HPLC. If Triton was detectable in the eluate, the capacity of the gel was reached.

Result:

The RP-18 gel binds 140 mg Triton X-100 / ml gel and the Amberchrome gel binds 160 mg Triton X-100 / ml gel.

Claims

claims

1. Therapeutically usable virus-inactivated albumin with an increased binding capacity for substances compared to pasteurization-inactivated albumin.

2. Albumin according to claim 1, wherein the binding capacity is increased by at least 10% compared to albumin virus-inactivated by pasteurization.

3. Albumin according to claim 1 and / or 2, wherein the binding capacity compared to pasteurized virus inactivated albumin is increased by at least 20 to 500%.

4. albumin according to at least one of claims 1 to 3, wherein the substances are low molecular weight active ingredients.

5. albumin according to at least one of claims 1 to 4, wherein the low molecular weight active substances are organic substances, nucleic acids, polypeptides having a molecular weight of at most 10,000 Da.

6. albumin according to at least one of claims 1 to 5 in liquid solution or in the solid state.

7. Albumin according to claim 6 in lyophilized form.

8. Process for the production of albumin, characterized by the combination of the following steps:

(b) Removal of the SD reagents by oil extraction and subsequent hydrophobic interaction chromatography, at least essentially, with a hydrophobic matrix for chromatography, in particular a matrix on which, if appropriate, hydrophobic Groups can be used, provided that these groups are aliphatic groups with C> 24, and a second albumin solution is obtained, which

(c) optionally one or more stabilizers from the group consisting of sugars, amino acids and sugar alcohols are added, with the proviso that no indole stabilizer and no C ₆ -C-fatty acid is used as the stabilizer, after which

(d) the second albumin solution, optionally mixed with a stabilizer, is finished and sterile filtered and, if appropriate, filled into final containers.

9. The method according to claim 8, characterized in that the virus inactivation is carried out at a temperature in the range from 25 to 40 ° C.

10. The method according to any one of claims 8 and / or 9, characterized in that the virus inactivation is carried out for a period in the range of 4 to 6 hours.

11. The method according to any one of claims 8 to 10, characterized in that glycine, glutamate, arginine, or lysine or a combination thereof is used as stabilizer.

12. The method according to any one of claims 8 to 10, characterized in that maltose and / or sorbitol is used as stabilizer.

13. The method according to any one of claims 8 to 12, characterized in that castor oil is used for the oil extraction.

14. The method according to any one of claims 8 to 13, characterized in that a polystyrene-divinylbenzene polymer or a methacrylate-based polymer is used as the hydrophobic matrix.

15. The method according to any one of claims 8 to 14, characterized in that branched or linear aliphatic groups with more than 24 carbon atoms are bound to the matrix.

16. The method according to any one of claims 8 to 15, characterized in that the albumin solution is deep-frozen after filling in the final containers.

17. The method according to any one of claims 8 to 16, characterized in that before or after steps (a), (b) or (c) any pre-kallikrein activator activity (PKA) present is removed in a manner known per se.

18. The method according to claim 17, characterized in that the albumin solution for removing the possibly present pre-kallikrein activator activity a) is brought into contact with activated carbon, after which the activated carbon is removed from the albumin solution, or b) is subjected to ion exchange chromatography.

19. The method according to claim 18, characterized in that step (A) is carried out at an albumin concentration between 1 and 25 wt .-%.

20. The method according to claim 19, characterized in that the albumin concentration is between 5 and 10 wt .-%.

21. The method according to claim 18, characterized in that step (B) is carried out at an albumin concentration between 5 and 10 wt .-%.

22. Procedure. according to one of claims 18 or 21, characterized in that the ion exchanger is an anion exchanger and the albumin solution is buffered with sodium acetate in the range of 100 - 150 mmol and the pH is in the range of 5.0 - 6.0.

23. The method according to claim 22, characterized in that the pH is <5.5.

24. The method according to any one of claims 18 or 21, characterized in that the ion exchanger is a cation exchanger and the albumin solution is buffered with sodium acetate in the range from 20 to 30 mmol and the pH is in the range from 4.8 to 6.0.

25. The method according to claim 24, characterized in that the pH is in the range of 4.8-5.2.

26. albumin solution obtainable by a process according to any one of claims 1 to 25.